Ngwakọta Mkpokọta RNA Mkpokọta Anụmanụ Ngụkọta Mwepụta RNA na Ngwa Nsacha maka anụ ahụ anụmanụ & cell
Nkọwa ngwa:
Ngwa ngwa na ịrụ ọrụ nke ọma wepụta mkpokọta RNA ịdị ọcha na ịdị elu dị elu site na anụ ahụ anụmanụ dị iche iche.
Ọ dịghị mkpa ichegbu onwe gị maka mbibi RNA.Sistemụ a niile enweghị RNase
Wepu DNA nke ọma site na iji kọlụm DNA- Cleaning
Wepu DNA na-agbakwunyeghị DNAse
Mfe-arụ ọrụ niile na-arụcha na ụlọ okpomọkụ
Ngwa ngwa-enwere ike ịrụcha ọrụ n'ime nkeji iri atọ
Safe — ọ dịghị organic reagent eji
Ịdị ọcha dị elu-OD260/280≈1.8-2.1
Nkọwa ngwa ngwa
Nkwadebe 50, 200 Preps
Ngwa a na-eji yaogho kọlụm na usoronke ụlọ ọrụ anyị mepụtara, nke nwere ike wepụ RNA dị elu na ịdị ọcha dị elu site na anụ ahụ dị iche iche nke anụ ọhịa na-arụ ọrụ dị elu. Ọ na-enye kọlụm DNA-Cleaning nke ọma, nke nwere ike ikewapụ ma gbasaa DNA genomic site na lysate na anụ ahụ, dị mfe ma na-azọpụta oge;Kọlụm naanị RNA nwere ike jikọta RNA nke ọma ma nwee ike hazie ya n'otu oge site na iji usoro pụrụ iche Ọtụtụ nlele.
Usoro dum bụ RNase-Free, nke mere na RNA amịpụtara adịghị emebi emebi;Buffer RW1, Buffer RW2 sistemu ịsacha ihe, nke mere na RNA enwetara enweghị protein, DNA, ion, na mmetọ ngwakọta organic.
Ngwa ngwa
| Ngwakọta Mkpokọta RNA anụmanụ | ||
| Ngwa ngwa | RE-03011 | RE-03014 |
| 50 T | 200 T | |
| Ihe nchekwa RL1* | 25ml | 100ml |
| Ihe nchekwa RL2 | 15ml | 60ml |
| Ihe nchekwa RW1* | 25ml | 100ml |
| Ihe nchekwa RW2 | 24ml | 96 ml |
| ddH2O na-efu RNase | 10ml | 40ml |
| RNA-naanị kọlụm | 50 | 200 |
| Kọlụm na-ehicha DNA | 50 | 200 |
| Akwụkwọ ntuziaka | 1 ibe | 1 ibe |
Ozi ngwaahịa
| Usoro | Kọlụm tụgharịa | Akụkụ ọcha | Kọlụm mbụ, reagent |
| Flux | 1-24 ihe atụ | Oge kwa nkwadebe | ~ 30 min (ihe atụ 24) |
| Centrifuge | Desktọpụ centrifuge | Pyrolysis nkewa | Nkewa nke Centrifugal |
| Ihe atụ | Anụ anụ;cell | Ọnụ ego nlele | Akpụkpọ anụ: 10-20 mg;Igwe: (1-5)×106 |
| Olu olu | 50-200 ml | Oke nbudata kacha | 850 μl |
Atụmatụ & uru
■ Ọ dịghị mkpa ichegbu onwe gị maka mbibi RNA;Sistemụ niile bụ RNase-Free
∎ Wepu DNA nke ọma site na iji kọlụm na-ehicha DNA
■ Wepu DNA na-agbakwunyeghị DNA
■ A na-arụ ọrụ niile dị mfe na ọnụ ụlọ okpomọkụ
■ Enwere ike ịrụcha ọrụ ngwa ngwa n'ime nkeji iri atọ
■ Nchekwa-ọ dịghị ihe reagent organic achọrọ
■ Ịdị ọcha dị elu -OD260 / 280≈1.8-2.1
Ngwa ngwa
Ọ dabara maka mmịpụta na ịdị ọcha nke mkpokọta RNA site na anụ ahụ dị ọhụrụ ma ọ bụ oyi kpọnwụrụ dị iche iche ma ọ bụ sel emebere.
Ngwa ngwaahịa
∎ Ngwa mgbada ala: njikọ cDNA nke mbụ, RT-PCR, cloning molecular, Northern Blot, wdg.
■ Ihe nlele: anụ ahụ anụmanụ, mkpụrụ ndụ omenala
■ Usoro onunu ogwu: Tissues 10-20mg, Sel (2-5) × 106
∎ Oke kachasị ike ijikọ DNA nke kọlụm nchacha: 80 μg
■ Olu olu: 50-200 μl
Eserese
Ngwakọta Mkpokọta RNA nke anụmanụ na-agwọ 20mg
Ihe nlele òké ọhụrụ, were 5% RNA 1% agar sachara ọcha
Glycogel electrophoresis
1: Spleen 2: Akụrụ
3: Imeju 4: Obi
Nchekwa na ndụ nchekwa
Enwere ike ịchekwa ngwa ahụ maka ọnwa 24 na okpomọkụ nke ụlọ (15-25 ℃) ma ọ bụ 2-8 ℃ maka ogologo oge.Enwere ike ịchekwa ihe nchekwa RL1 na 4 ℃ maka otu ọnwa ka ịgbakwunye β-mercaptoethanol (nhọrọ).
Edemede ndị edepụtara
1.Ọ BỤRỤ: 18.808:Zheng, Q., Qin, F., Luo, R., et al.Lipid-dị ka nanoparticles mRNA-kwadoro maka ndezi imeju site na njikarịcha nke imepụta ihe mejupụtara etiti.Adv.ỌrụMater.2021, 31, 2011068.doi:10.1002/adfm.202011068.
2.Ọ BỤRỤ: 18.187:He X, Hong W, Yang J, et al.Apoptosis nke mkpụrụ ndụ n'otu n'otu na nkwadebe sel sel ọgwụgwọ na-arụ ọrụ immunomodulatory site na ntọhapụ nke phosphatidylserine.Ebumnuche Ntugharị Mgbaama Ebe ahụ.2021 Jul 14;6 (1):270 .doi: 10.1038/s41392-021-00688-z.
3.Ọ BỤRỤ: 17.97: Dai Z, Liu H, Liao J, et al.N7-Methylguanosine tRNA mgbanwe na-akwalite ntụgharị asụsụ mRNA oncogenic ma na-akwalite ọganihu intrahepatic cholangiocarcinoma.Mkpụrụ ndụ Mol2021 Jul 29:S1097-2765(21)00555-4.doi: 10.1016/j.molcel.2021.07.003.
4.Ọ BỤRỤ: 9.225: Cao X, Shu Y, Chen Y, et al.Mettl14-Mediated m6A Mgbanwe na-eme ka mmeghari imeju dị mma site na ijikwa Endoplasmic Reticulum Homeostasis.Cell Mol Gastroenterol Hepatol.2021;12 (2): 633-651 .doi: 10.1016/j.jcmgh.2021.04.001.
Ngwa iche iche RNA maka isi mmalite sampledị:
Cell, osisi, viral, ọbara, wdg.
A naghị ewepụta RNA ma ọ bụ RNA dị ala
A na-enwekarị ihe dị iche iche na-emetụta arụmọrụ mgbake, dịka: ọdịnaya RNA anụ ahụ, usoro ọrụ, elution volume, wdg.
1. Ice bath ma ọ bụ cryogenic (4 Celsius C) centrifugation mere n'oge ọrụ.
Nkwanye: Na-arụ ọrụ na ụlọ okpomọkụ (15-25 Celsius C) n'ime usoro dum, emela ice bat na centrifuge na obere okpomọkụ.
2. Nchekwa nlele na-ezighi ezi ma ọ bụ oge nchekwa oke.
Nkwanye: Chekwaa ihe nlele na -80 Celsius ma ọ bụ ifriizi na mmiri mmiri nitrogen ma zere iji friza eme ihe ugboro ugboro;gbalịa iji anụ ahụ ọhụrụ ma ọ bụ mkpụrụ ndụ emebere maka mmịpụta RNA.
3. ezughị ezu sample lysis.
Nkwanye: Mgbe ị na-eme ka anụ ahụ dị n'otu, hụ na anụ ahụ na-emekọ ihe nke ọma yana na mkpụrụ ndụ anụ ahụ kewara nke ọma iji kọwaa ntọhapụ nke RNA.
4. A naghị atụkwasị eluent nke ọma.
Nkwanye: Kwenye na ddH enweghị RNase2A na-agbakwunye O n'ime ala n'etiti akpụkpọ ahụ kọlụm nke ịdị ọcha.
5. Agbakwunyeghị olu ziri ezi nke ethanol zuru oke na Buffer RL2 ma ọ bụ Buffer RW2.
Nkwanye: Soro ntuziaka a, tinye nha ziri ezi nke ethanol zuru oke na Buffer RL2 na Buffer RW2 wee gwakọta nke ọma tupu i jiri ngwa ahụ.
6. Usoro ihe nlele anụ ahụ adịghị mma.
Nkwanye: Jiri 10-20 mg nke anụ ahụ ma ọ bụ (1-5) × 106mkpụrụ ndụ kwa 500 μl ihe nchekwa RL1, n'ihi na iji anụ ahụ gabigara ókè nwere ike ime ka mmịpụta RNA belata.
7. Olu elution na-ezighi ezi ma ọ bụ elution ezughị ezu.
Nkwanye: Oke elution nke kọlụm nchacha bụ 50-200 μl;Ọ bụrụ na mmetụta elution adịghị afọ ojuju, a na-atụ aro ịgbatị oge ntinye ụlọ okpomọkụ mgbe agbakwunyere ddH na-enweghị ọkụ RNase.2O, ọmụmaatụ maka nkeji 5-10.
8.The kọlụm dị ọcha nwere ethanol fọdụrụnụ mgbe Buffer RW2 saa.
Nkwanye: Ọ bụrụ na e nwere ihe fọdụrụ na ethanol mgbe ịsachara Buffer RW2, centrifugation tube efu maka 1min, enwere ike ịbawanye oge ọrụ centrifugation tube efu ruo 2min, ma ọ bụ enwere ike idobe kọlụm dị ọcha na ụlọ okpomọkụ maka 5 min iji wepụ ethanol fọdụrụnụ nke ọma.
RNA dị ọcha na-eweda ya ala
Àgwà nke RNA a sachara ejikọta na ihe ndị dị ka ichekwa ihe nlele, mmetọ RNase, na nhazi, wdg.
1. A naghị edebe ihe nlele anụ ahụ n'oge.
Nkwanye: Ọ bụrụ na ejighị ihe nlele anụ ahụ ma ọ bụ sel mee ihe n'oge kwesịrị ekwesị mgbe nchịkọtachara, ozugbo cryopreserve na -80 Celsius C ma ọ bụ nitrogen mmiri mmiri.Iji wepụ RNA, jiri anụ ahụ ma ọ bụ ihe nlele cell ewepụtara ọhụrụ mgbe ọ bụla enwere ike.
2. Ugboro ugboro ifriizi-thawing nke anụ ahụ sample.
Nkwanye: Mgbe ị na-echekwa ihe nlele anụ ahụ, ọ kacha mma ịkpụ ya n'ime obere iberibe maka nchekwa, ma wepụ otu n'ime iberibe ahụ mgbe ị na-eji ha ka ịzenarị oyiri ugboro ugboro nke ihe nlele ahụ na mmebi nke RNA.
3. A na-ewebata RNase ma ọ bụ ghara iyi uwe mkpuchi, ihe mkpuchi, wdg n'oge ọrụ ahụ.
Nkwanye: A na-eme nnwale mmịpụta RNA kacha mma n'ime ụlọ njikwa RNA dị iche iche wee kpochapụ tebụl tupu nnwale ahụ.
Yiri gloves na masks enwere ike iwepụ n'oge nnwale ahụ iji belata mmebi RNA kpatara site na iwebata RNase.
4. A na-eji RNase metọọ reagents n'oge eji.
Nkwanye: Dochie jiri ngwa Mkpokọta anụ ọhịa RNA ọhụrụ maka nnwale ndị metụtara ya.
5. Ọkpụkpọ centrifuge, ndụmọdụ, wdg eji eme ihe na RNA na-emetọ ya na RNase.
Nkwanye: Kwenye na tubes centrifuge, ndụmọdụ, pipettes, wdg. ejiri na mmịpụta RNA bụ RNase niile.
RNA emepụtara dị ọcha na-emetụta nnwale ndị dị n'okpuru ala
RNA sachara site na kọlụm dị ọcha, ọ bụrụ na ion nnu, ọdịnaya protein buru ibu ga-emetụta nnwale nke ala, dị ka: ntụgharị ntụgharị, Northern Blot et al.
1. RNA nke elutioned nwere residues ion nnu.
Nkwanye: Kwenye na agbakwunyere oke ethanol ziri ezi na Buffer RW2 ma mee 2 kọlụm nchacha ọcha na ọsọ centrifugal nke egosiri maka ịrụ ọrụ;Ọ bụrụ na enwere ihe fọdụrụ ion nnu, hapụ kọlụm nke ịdị ọcha na Buffer RW2 maka 5 min na ụlọ okpomọkụ ma mee centrifugation iji bulie mwepụ nke mmetọ nnu.
2. Ethanol fọdụrụnụ na elutioned RNA.
Nkwanye: Kwenye na mgbe ịsachara RW2 na-enweghị ihe ọ bụla, rụọ ọrụ centrifugation tube efu na ọsọ centrifugation egosipụtara maka ịrụ ọrụ, mụbaa oge ọrụ centrifugation tube efu ruo 2 min ma ọ bụrụ na ọ ka nwere ihe fọdụrụ na ethanol, ma ọ bụ hapụ ya n'ime ụlọ okpomọkụ maka 5 min mgbe centrifugation efu gachara iji bulie mwepụ nke ethanolue.
